Self-sealing MEMS spray-nozzles to prevent bacterial contamination of portable inhalers for aqueous drug delivery

Pulmonary drug delivery by portable inhalers is the gold standard in lung disease therapy. An increasing focus on environmentally friendly inhalation currently spurs the development of propellant-free devices. However, the absence of propellants in the drug creates a need for suitable sealing systems that can ensure the pathogenic safety of devices. Traditionally, liquid drug inhalers incorporate a spray nozzle and a separate check valve. Here we show a fully integrated MEMS-based spray system for aqueous drug solutions and demonstrate its bacterial safety. The device comprises a thin silicon membrane with spray orifices, which self-seal against a compliant parylene valve seat underneath. This sealing system prevents bacterial ingrowth in its default closed state, while actuation lifts the membrane from the valve seat upon pressurization and sprays an inhalable aerosol from the nozzles. To seal against bacterial contamination effectively, we found that a contact force between the valve seat and the membrane (featuring the spray nozzles) is needed. In our testing, both self-sealing and an otherwise identical unvalved version of the spray chip can be bacterially safe in continued use when thoroughly cleaned of excess fluids and subjected to low bacterial loads for brief periods. However, when directly exposed to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$10^{7}$$\end{document}107 CFU/ml of our test organism Citrobacter rodentium for 24 h, unvalved systems become contaminated in nearly 90% of cases. In contrast, self-sealing spray chips reduced contamination probability by 70%. This development may enable preservative-free drug formulations in portable inhalers that use propellant-free aqueous drug solutions. Supplementary Information The online version contains supplementary material available at 10.1007/s10544-022-00628-w.


S Aggregated data on bacteria found on inhalation devices
Most devices analyzed in the following studies are stationary inhalers, such as nebulizers. Some of the studies were performed in a clinical setting, where inhalation devices are subject to sterilization procedures. Surface contamination of portable inhalation devices is rather common as shown by Levesque and Johnson ( ), who analyzed the portable inhalers of children and found all mouthpieces of the analyzed inhalers positive for bacterial growth.
% of these inhalers were never washed. The cultured organisms of this study were not speciated. In a study performed by Barnes et al ( ), the stationary home nebulizers of patients were investigated, most of which showed severe pathogenic contamination. Interestingly, this is the only study in which gram-positive organisms dominated the collected bacteria. A study performed by Borovina et al ( ) was conducted in in Australia on a total of pMDIs. % of these devices showed visible debris on the inside of the mouthpiece, and % of the devices were colonized with microorganisms. Some of the collected bacteria were shown to be antibiotically resistant. The most recent study by Jarvis et al ( ) concluded that even recommended nebulizer washing techniques might not eradicate microorganisms on inhalation devices, but only found % of the analyzed inhalers to be colonized. Bacteria collected from inhalation devices are aggregated in

S Raw maximum membrane deflection data
We have measured membrane deflection for three different membrane sizes, which have all come from the same wafer that our tested chips originate from, using a Wyko NT optical profilometer. CF chips have a membrane with an intentionally deflected membrane (by the Parylene gasket) of . µm. This leads to an initial force that is acting between valve seat and membrane at all times, even when no pressure is applied to the chip. The NF chips should show no membrane deflection other than residual deflection due to compressive stress. For the NF chip design we measure deflections at rest in the order of -nm. For the CF chips we measure values close to . µm, the height of the Parylene gasket between valve seat and membrane. For the µm membranes we measure slightly smaller maximum deflection values, possibly due to Parylene compression.
Table contains the deflection data for the NF and CF chips, measured on samples that originate from the same wafer, using white light interferometry.

S Dynamic ingrowth supplement
A total of spray chips were subjected to dynamic ingrowth testing, out of which four were removed from the test due to a broken membrane and five more due to a leaking Luer connection. In total, % of the spray chips made it through the whole cycle of six actuations. A detailed illustration of how many chips made it through the different steps is given in Fig. , which shows the testing procedure for dynamic ingrowth testing.

S . Raw dynamic ingrowth data
The raw optical density data for the dynamic ingrowth test is given in

S Static ingrowth supplement
We removed packages that showed leakage (either around the glue connection of the chip or the Luer connection

Fig.
Static ingrowth protocol, with the number of chips at each step given in brackets.
to the syringe) and were therefore prone to bacteria contaminating the syringe without passing the sealing system. We further removed packages with damaged membranes after the autoclavation step. In total, out of chips were sorted out before going through the h ingrowth procedure. Further, a single chip was sorted out post-experiment as no bacteria could be cultivated from its bacterial well. Fig. shows the testing procedure for static ingrowth testing.
While performing the ingrowth test we encountered several reasons for spray chips to fail the ingrowth test that do not bear on the validity of the spray chips themselves.
-Membrane damaged during autoclavation of spray packages. -Test package leaks from the Luer connection.
-Test package leaks from the glue seam around the spray chip.  Table shows the raw data of collected optical densities from syringe packages. Testing was performed over two months with separate testing runs. In total packages were tested, all chips had a membrane width of µm We further collected the bacterial density in the bacterial microwells which are loacted on top of the spray chip. This measurement serves two functions: first, we assure that bacteria do survive for the time of the ingrowth test in the microwell. Second, this indicated just how many bacteria A single sample out of samples was excluded from the dataset, as no bacteria could be cultivated from the bacterial well. This data is collected in Table ??.

S CFU calculation from OD
The number of colony-forming unit (CFU) that are present in a growth medium based on measured optical density (OD) were determined using plating at different Citrobacter rodentium (ICC ) OD. OD was acquired from ml samples, collected into -well plates. Equation is valid in an optical density range between 0.07 < < 1.5.
a) Glued packages, where the spray chip is glued into the nozzle holder and the microwell is glued on top. Looking from the top, the black rectangle in the middle is the spray chip, b) from left to right shows the syringe with the Luer connection, then the nozzle package, the microwell and the lid, c) shows the microwell filled with a bacterial solution after removing the Parafilm cover from the syringe, which is visualized in d).

Table
Raw optical density data for the dynamic ingrowth test.